Hematopoietic cell transplantation (HCT) can cure a variety of benign and malignant hematopoietic disorders, but graft-versus-host disease (GVHD) remains the primary case of transplant-related morbidity, disability and mortality, thereby limiting the use of HCT. A dominant mechanism of tolerance mediated by regulatory T cells (Tregs) has obvious therapeutic implications in preventing GVHD, as they can control GVHD and may spare GVL effects depending on their TCR specificity. In an attempt to apply Tregs in clinical HCT, current approaches are focused on adoptive transfer of polyclonal, ex vivo expanded, naturally derived Tregs (nTregs) into transplant recipients before or after HCT. However, these polyclonal nTregs are expected to have a low potency in controlling GVHD and produce non-selective immune suppression without discriminating for GVHD and GVL reactions. The current proposal is aimed at increasing the potency and specificity of Treg therapy by using alloreactive Tregs. Our long-term goal is to using alloantigen-reactive Tregs to prevent GVHD while preserving the GVL effect after HCT in humans. The objective of this proposal is to achieve this goal in pre-clinical bone marrow transplantation (BMT) models in mice. Our central hypothesis is that allo-specific Tregs are highly efficient in the prevention of GVHD, and that Tregs specific for mHAgs expressed by epithelial tissues but not by malignant cells may control GVHD while preserving GVL activity. We plan to test our hypothesis and accomplish the objective by pursuing 3 specific aims: 1) To evaluate the potency of alloantigen-specific Tregs in the prevention of GVHD; 2) To determine the effect of cognized antigen-distribution in the recipient on Treg-mediated GVHD; and 3) To define the effect of allo-specific Tregs in GVHD and GVL activity. The information obtained from this proposal is expected to establish a new strategy to prevent GVHD while sparing GVL effect by conferring Tregs with certain antigen specificities and applying them to protect GVHD target tissues but not tumor from attack by donor T cells.